distribution of microbial arsenic reduction, oxidation and extrusion

Distribution of Microbial Arsenic Reduction, Oxidationand Extrusion Genes along a Wide Range ofEnvironmental Arsenic ConcentrationsLorena V. Escudero1,2, Emilio O. Casamayor4, Guillermo Chong3, Carles Pedros-Alio5,

Cecilia Demergasso1,2*

1 Centro de Investigacion Cientıfico Tecnologico para la Minerıa-CICITEM, Universidad Catolica del Norte, Antofagasta, Chile, 2 Centro de Biotecnologıa, Universidad

Catolica del Norte, Antofagasta, Chile, 3 Departamento de Ciencias Geologicas, Universidad Catolica del Norte, Antofagasta, Chile, 4 Biogeodynamics & Biodiversity Group,

Centre d’Estudis Avancats de Blanes, CEAB-CSIC, Blanes, Spain, 5 Institut de Ciencies del Mar, CSIC, Barcelona, Spain

Abstract

The presence of the arsenic oxidation, reduction, and extrusion genes arsC, arrA, aioA, and acr3 was explored in a range ofnatural environments in northern Chile, with arsenic concentrations spanning six orders of magnitude. A combination ofprimers from the literature and newly designed primers were used to explore the presence of the arsC gene, coding for thereduction of As (V) to As (III) in one of the most common detoxification mechanisms. Enterobacterial related arsC genesappeared only in the environments with the lowest As concentration, while Firmicutes-like genes were present throughoutthe range of As concentrations. The arrA gene, involved in anaerobic respiration using As (V) as electron acceptor, wasfound in all the systems studied. The As (III) oxidation gene aioA and the As (III) transport gene acr3 were tracked with twoprimer sets each and they were also found to be spread through the As concentration gradient. Sediment samples had ahigher number of arsenic related genes than water samples. Considering the results of the bacterial communitycomposition available for these samples, the higher microbial phylogenetic diversity of microbes inhabiting the sedimentsmay explain the increased number of genetic resources found to cope with arsenic. Overall, the environmental distributionof arsenic related genes suggests that the occurrence of different ArsC families provides different degrees of protectionagainst arsenic as previously described in laboratory strains, and that the glutaredoxin (Grx)-linked arsenate reductasesrelated to Enterobacteria do not confer enough arsenic resistance to live above certain levels of As concentrations.

Citation: Escudero LV, Casamayor EO, Chong G, Pedros-Alio C, Demergasso C (2013) Distribution of Microbial Arsenic Reduction, Oxidation and Extrusion Genesalong a Wide Range of Environmental Arsenic Concentrations. PLoS ONE 8(10): e78890. doi:10.1371/journal.pone.0078890

Editor: Michiel Vos, University of Exeter Medical School, United Kingdom

Received January 23, 2013; Accepted September 17, 2013; Published October 31, 2013

Copyright: � 2013 Escudero et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This research was funded by grant BIOARSENICO from Fundacion BBVA, Spain, and the Chilean government through FONDECYT Project 1100795 fromthe Science and Technology Chilean Commission (CONICYT). LVE was supported by a fellowship from CICITEM, Antofagasta, Chile, and EOC by DARKNESSCGL2012-32747, from the Spanish MINECO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of themanuscript.

Competing Interests: The authors have declared that no competing interest exist.

* E-mail: [email protected]

Introduction

Arsenic toxicity is a major problem in many parts of the world

where drinking water supplies are heavily contaminated [1,2].

This has prompted considerable research on biogeochemical and

microbiological processes that control the distribution and

mobilization of As in aquatic environments [3,4]. Despite the fact

that arsenic transformations are carried out by a large variety of

microorganisms, the number of different genes involved seems to

be limited to a few types that are widely distributed across

phylogenetic lineages. Therefore, there is a possibility of detecting

the presence of such genes in the environment with a limited

number of primers [5,6]. This possibility is attractive because the

monitoring of the presence of As-related genes may help in

detecting and managing As contamination events.

The genes used by prokaryotes to cope with As fit into five

groups: (i) As(V) detoxifying reduction ars operon, (ii) As(V) arr

respiratory reduction operon, (iii) As(III) oxidation aio operon, (iv)

As(III) extrusion genes acr3, and (v) methylation genes not specific

for arsenic [7,8]. Many bacteria can detoxify As by the Ars system

—plasmid or chromosome encoded—, which is widespread in

nature and has been extensively studied [9,10]. The key enzyme is

the arsC product that reduces As(V) to As(III). The latter can then

be extruded out of the cell by the pump coded by arsB. The operon

includes up to five genes, some of them are regulators and the arsC

gene is almost always present [11]. Three different ArsC

prokaryotic families have been defined based on the protein

structures, the reduction mechanisms, and the location of the

catalytic cysteine residues: i) the glutathione (GSH)/glutaredoxin

(Grx)-coupled class) (plasmid R773 of Escherichia coli) [12], ii) the

thioredoxin (Trx)/thioredoxin reductase (TrxR)-dependent class

(plasmid pl258 of Staphylococcus aureus) [13] and iii) the mycothiol

(MSH)/mycoredoxin (Mrx)-dependent class found in Actinobac-

teria [14–16]. Kinetics data of arsenate reduction have shown a

higher catalytic efficiency in thyoredoxin (Trx)- than in glutar-

edoxin (Grx)-linked arsenate reductases [16]. Some authors have

suggested that these families arose independently [7,12]. Con-

versely, a single early origin of the arsC gene and subsequent

sequence divergence has also been proposed [17]. In any case, the

families are sufficiently different that they require different primer

PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e78890

sets to be fully targeted. Thus, besides the three sets of primers

described in the literature [18–20], here we developed three

additional sets to cover the full genetic diversity of the arsC gene.

Another widespread set of genes involved in As reduction is the

arr operon (As respiration). The arr and ars operons are not

mutually exclusive and the presence of both has been shown in

Shewanella sp. [18]. Bacteria with the arr operon use As(V) as

electron acceptor and reduce it to As(III), a more toxic and more

mobile form [18]. Some bacteria, however, respire both As(V) and

sulfate, precipitating As sulfides such as realgar and orpiment, thus

providing a potential bioremediation mechanism [21]. Arsenic

sulfide-precipitating microorganisms have been found in several

contaminated environments in northern Chile by means of most

probable number estimations and isolations in pure culture [22].

The functional gene for arsenate respiration, arrA, is highly

conserved [7]. Three primer sets have been designed in the

literature [6,23,24] and they were all tested in the present work.

The microbial oxidation of arsenite is also a widespread

mechanism in soil and water systems containing As. Many

bacteria oxidize arsenite as a step in detoxification, since As(V)

is less toxic than As(III). But some bacteria are able to use As(III) as

a source of electrons for respiration. Recently, a common

nomenclature has been proposed for homologous arsenite

oxidizing genes [25]. Thus, for example, aioA (arsenite oxidation)

stands for the formerly named aoxB, asoA and aroA. The genes for

aerobic arsenite oxidation are widely distributed in the bacterial

domain and two different primer sets have been designed in the

literature, which were used here to detect these genes [5]. In

addition, a new gene family, arxA, has been identified through

mutagenesis and physiological experiments that is required for

chemoautotrophic growth with arsenite coupled to nitrate

respiration [26].

The acr3 gene product is a pump that extrudes As(III) out of the

cells [27,28]. The function of this protein is analogous to that of

arsB, a component of the more common detoxifying operon. Two

families have been identified and the corresponding primers have

been published [29] and were used here to detect their presence.

Finally, methylation has been proposed as an additional

detoxification strategy [30]. An arsenite S-adenosylmethionine

methyltransferase (arsM) has been characterized in Rhodopseudomo-

nas palustris and belongs to the UbiE/Coq5 C-methyltransferase

family [31]. The arsM gene has been detected in more than 120

Bacteria and in about 15 Archaea [32]. However, the enzyme is

not specific for the As(V) to As(III) reduction step [7] and,

therefore, we have excluded this gene from the present study.

Most of the primers mentioned above have not been tested in

natural environments, or have been tested in one or a few natural

samples only [5,19,24,33], covering a very limited range of As

concentrations. In addition, some of the primers do not target the

whole diversity of known genes [19,20,34]. In the Andean

altiplano (including areas of Chile, Argentina, Bolivia and Peru),

As contamination is a natural process that has occurred for long

periods of time. Many people drink water with toxic As levels, even

up to 0.6 mg L21 [35]. Some environments in this region show the

largest As concentrations recorded in natural systems [22]. We,

therefore, tested 11 [5,6,18–20,23,24,29] of the 19 available

primers [26,36–40] in a series of environments including thermal

springs, stream waters, lagoons, mats lining hot springs and several

types of sediments that, besides being conveniently close in space,

offered a range of As concentrations spanning six orders of

magnitude.

Materials and Methods

Site descriptionTable 1 shows a summary of the geographical location, altitude

and other environmental parameters for the systems studied. We

chose different As-rich environments located between UTM 19 K

572171 and 603405 South and between 7427145 and 7622927

West (Fig. 1 A and B). Most sites were above 3700 m of altitude.

These included several samples from the main Andean salts

deposits (Salar de Ascotan), from a geothermal geyser field (El

Tatio) and from incoming rivers (i.e. ‘‘quebradas’’) to Salar de

Atacama (Quebrada de Jere and Quebrada Aguas Blancas). No

specific permissions were required for sampling in Salar de

Ascotan, Quebrada de Jere, Quebrada Blanca and El Tatio. For

sampling in El Tatio we informed the Comunidad Indıgena de

Toconce and Socaire. All these systems shared the presence of As

in large or small concentrations (see Table 1). Salar de Ascotan

was studied in more detail because the As concentrations were the

highest found in the area. Salar de Ascotan is an athalassohaline

environment located at the bottom of a tectonic basin surrounded

by volcanic systems in east-west direction, including some active

volcanoes over 5000 m high, with the highest altitudes close to

6000 m. Climate is characterized by large daily thermal oscilla-

tions. High solar irradiance, and strong and variable winds cause

intense evaporation (about 4.5 mm day21) while precipitation is

about 120 mm year21 [41]. Water inputs include surface drainage

from the snow fields on volcanoes, ground waters with a strong

geothermal component and spring waters commonly reaching

23uC to 25uC. The saline crusts are mainly composed of chlorides

(halite) and sulfates (gypsum) with important borate ore deposits

composed mostly of ulexite with significant amounts of As sulfide

minerals. This system exhibits high spatial and temporal variability

with water salinities ranging from freshwater to salt-saturated

brines [42].

Sample collection and processingFour sampling expeditions to Salar de Ascotan were conducted

in November 2004, August 2005, June 2006 and April 2007

(Table 1). Eighteen sampling sites were selected within the basin

(P1 to P11) and three thermal springs (‘‘vertientes’’ V4, V6 and

V10) for sampling both water and sediments at different times

(Table 1 and Fig. 1 A and B). Overall, 29 samples were analyzed.

Temperature and pH were measured with a pH meter Orion

model 290. A conductivity meter Orion model 115 was used for

measuring salinity, conductivity and total dissolved solids. Oxygen

was measured with a Thermo Orion model 9708. Water samples

were kept in polyethylene 2-L bottles in an icebox until further

processing during the next 24 hr. Samples for total cell counts were

fixed in situ with 4% formaldehyde (vol/vol, final concentration)

overnight at 4uC. Counts were done by epifluorescence micros-

copy staining with a DNA-specific dye, 49, 6-diamidino-2-

phenylindole (DAPI) with a Leica DMLS epifluorescence micro-

scope. Arsenic concentrations were measured using Hydride

generation atomic absorption spectroscopy (HG-AAS), directly

on the brine samples and after acid digestion of the sediment

samples [22].

Total DNA extractionDNA collection and extraction were done as described before

[22]. Between 800 and 1000 mL of water was filtered through

0.2 mm polycarbonate membranes (Nuclepore). The filters were

stored at 220uC in 1 mL lysis buffer (50 mM Tris–HCl pH = 8.3,

40 mM EDTA and 0.75 M sucrose) [43]. For sediment samples,

nucleic acids were extracted from 25 to 50 g sediment (wet

Distribution Environmental of Arsenic Genes


Ta

ble

1.

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79

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sco

tan

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ne

sed

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nt

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09

15

45

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23

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nB

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ese

dim

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21

71

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tan

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ng

sed

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nt

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sco

tan

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ne

sed

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nt

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03

55

73

66

93

74

81

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ota

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ese

dim

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29

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36

79

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39

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P7

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nB

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ese

dim

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60

90

35

57

77

70

37

48

65

04

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sco

tan

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weight), and were actively vortexed in a salt solution (PBS buffer

1x, Tween 20 at 10% v/v) at 120 rpm. The supernatant was

filtered and processed as previously done with water samples. The

filters were incubated with lysozyme and proteinase K [43], and

genomic DNA was extracted with a High Pure Template

Preparation Kit (Qiagen).

Figure 1. Maps of the sampling location. (A) Map of northern Chile showing the areas where samples were taken: 24 in Ascotan, three in El Tatio,and 2 in Atacama. (B) Modified satellite image of Salar de Ascotan showing the location of sampling spots. The contours of As level in water wasconstructed using the Surfer software program (v.7.0, Golden Software, USA). Point P6 had the highest concentration in the water. Concentrations insediments were higher in all cases.doi:10.1371/journal.pone.0078890.g001

Table 2. Primer sets used in this study for PCR amplification of several genes involved in the arsenic cycle.

Targeted gene Primer Set1 Primer name2 Primer sequence (59 – 39)Ampliconlenght (bp)

Reference (systemstudied)

Arsenate respiratoryreductase

arrA 1 arrAf AAG GTG TAT GGA ATA AAG CGT TTG TBG GHG AYT T 160–200 [6]

arrAr CCT GTG ATT TCA GGT GCC CAY TY V GGN GT (Haiwee Reservoir andShewanella sp. ANA-3)

arrA 2 AS1f CGA AGT TCG TCC CGA THA CNT GG 625 [24]

AS1r GGG GTG CGG TCY TTN ARY TC (Cambodian Sediments)

AS2f (nested) GTC CCN ATB ASN TGG GAN RAR GCN MT

arrA 3 HAArrA-D1f CCG CTA CTA CAC CGA GGG CWW YTG GGR NTA 500 [23]

HAArrA-G2r CGT GCG GTC CTT GAG CTC NWD RTT CCA CC (Mono Lake and SearlesLake, California)

Arsenate reductase arsC-Grx-Sun mix amlt-42-f TCG CGT AAT ACG CTG GAG AT 334 [19]

amlt-376-r ACT TTC TCG CCG TCT TCC TT (Bacterial strain andplasmid)

smrc-42-f TCA CGC AAT ACC CTT GAA ATG ATC

smrc-376-r ACC TTT TCA CCG TCC TCT TTC GT

arsC-Grx-Saltikov Q-arsC-f1 GAT TTA CCA TAA TCC GGC CTG T 200–300 [18]

Q-arsC-r1 GGC GTC TCA AGG TAG AGG ATA A (Shewanella sp. strainANA-3)

arsC-Trx-Villegas arsCGP-Fw TGC TG ATTT AGT TGT TAC GC 353 [20](As-resistant bacteriaisolated fromcontaminated soil)

arsCGP-Rv TTC CTT CAA CCT ATT CCC TA

arsC-Trx1a arsC 4f TCH TGY CGH AGY CAA ATG GCH GAA G 300–400 This study

arsC 4r GCN GGA TCV TCR AAW CCC CAR TG

arsC-Trx1b arsC 5f GGH AAY TCH TGY CGN AGY CAA ATG GC 300–400 This study

arsC 5r GCN GGA TCV TCR AAW CCC CAR NWC

arsC-Trx2 arsC 6f CAC VTG CMG RAA DGC RAR RVV DTG GCTCG 300–400 This study

arsC 6r1 YKY CRY CBR YVA DRA TCG G

arsC 6r2 TTR WAS CCN ACG WTA ACA KKH YYK YC

arsC 6r3 YYV HWY TSK TST TCR YKR AAS CC

Arsenite transporter acr3 1 dacr1-F GCC ATC GGC CTG ATC GTN ATG ATG TAY CC 750 [29]

dacr1-R CGG CGA TGG CCA GCT CYA AYT TY TT

acr3 2 dacr5-F TGA TCT GGG TCA TGA TCT TCC CVA TGM TGV T 750

dacr4-R CGG CCA CGG CCA GYT CRA ARA ART T

Arsenite oxidase aioA 1 aroA #1F GTS GGB TGY GGM TAY CAB GYC TA 500 [5](Arsenic-impacted soils,

aroA #1R TTG TAS GCB GGN CGR TTR TGR AT

aioA 2 aroA #2F GTC GGY YGY GGM TAY CAY GYY TA 500 sediments and

aroA #2R YTC DGA RTT GTA GGC YGG BCG geothermal mats, USA)

1Code used in the present paper.2Name used in the original publication.doi:10.1371/journal.pone.0078890.t002



Detoxifying arsenate reductase arsC genesThe arsC gene was targeted using six primer sets (Table 2). The

sequences targeted by each primer set are shown in Figure 2. The

first primer set (arsC-Grx-Sun) contained a mixture of primers

amlt-42-f/amlt-376-r and smrc-42-f/smrc-376-r [19]. The PCR

amplification conditions were as follows: 95uC for 3 min, 40 cycles

of 95uC for 15 s denaturation, 60uC for 15 s annealing, and 72uCfor 15 s elongation, in a primer mixture of 0.25 mM each. These

primers amplify a fragment of 353 bp of the arsC gene. The second

set (arsC-Grx-Saltikov) consisted of primers Q-arsC-f1 and Q-

arsC-r1 [18]. The PCR amplification conditions were as follows:

95uC for 10 min, 40 cycles of 95uC for 30 s (denaturation), 60uCfor 1 min (annealing), and 72uC for 30 s (elongation), at 0.5 mM

each primer. The third set (arsC-Trx-Villegas) consisted of primers

arsCGP-Fw and arsCGP-Rv [20] for the amplification of the ArsC

thioredoxin dependant mechanism (arsC-Trx). The PCR program

for these primers was 5 min denaturation at 95uC followed by 35

cycles of 95uC for 90 s, 48uC for 90 s, and 72uC for 2 min, and

finally 5 min extension at 72uC. The concentration of primers was

0.3 mM [20].

The next three sets of primers (arsC-Trx1a, arsC-Trx1b, arsC-

Trx2) were designed in the present study. We collected the known

arsC sequences from the literature [12] and from GenBank

(retrieved from NCBI) and we also searched the available genomes

of the predominant phylotypes in the studied environment. It

appeared that Proteobacterial sequences were well covered by the

arsC-Grx-Sun [19]. Some Firmicutes sequences, on the other

hand, were not covered by any primer set. Sequences were aligned

using Clustal X [44] and manually corrected. The aligned arsC

sequences were used to construct a baseline phylogenetic tree

using neighbor-joining (NJ), maximum parsimony (MP) and

maximum likelihood (ML) methods (Figure 2). The tree was

constructed using the Molecular Evolutionary Genetic Analysis

(MEGA) 4 (http://www.megasoftware.net) software with 500

bootstrap replicates. To design the arsC-Trx1a and arsC-Trx1b

sets of primers we used arsC sequences from the following bacteria:

Bacillus thuringiensis, Bacillus cereus, Geobacillus kaustophilus, Bacillus

clausii, Oceanobacillus iheyensis and Bacillus halodurans (Fig. 2 and Fig.

S1 A). The sequences used for designing primer set arsC-Trx2

were Geobacillus thermoglucosidasius, Geobacillus sp., Alkaliphilus orem-

landii OhILAs and Bacillus subtilis (Fig. 2 and Fig. S1 B). The

Sequence Manipulation Suite (SMS) (http://bioinformatics.org/

sms2/index.html) was used to verify the properties of each

designed primer.

Primers arsC-Trx1a and arsC-Trx1b amplify a 300–400 bp

fragment of the arsenate reductase gene (Table 2). The PCR

conditions with the arsC-Trx1a set were 5 min denaturation at

95uC, followed by 35 cycles of 95uC for 90 s, 46.7uC for 90 s, and

72uC for 3 min, and finally 5 min extension at 72uC. The PCR

conditions with the arsC-Trx1b set were the same as for arsC-

Trx1a set but using a different annealing temperature (60uC). For

the primer set arsC-Trx2 we tried one forward primer and three

reverse primers. The reverse primer arsC6r2 resulted in the best

outcome. The PCR conditions were 5 min denaturation at 95uC,

followed by 35 cycles of 95uC for 90 s, 54.5uC for 90 s, and 72uCfor 3 min, and finally 5 min extension at 72uC. The concentration

of primer was 0.3 mM. The PCR representative products (14) were

purified using a QIAGEN PCR cleanup kit according to

manufacturer instructions and sent for sequencing to Macrogen

(Macrogen Inc., Korea, www.macrogen.com) to confirm that the

amplicon obtained with the newly designed primers were bona

fide arsC gene fragments. Sequences for the arsC genes were sent to

BLAST (Basic Local Alignment Search Tool) algorithm [45] at the

National Center for Biotechnology Information (NCBI, http://

www.ncbi.nlm.nih.gov/BLAST/) for BLASTX and BLASTN

searches to determine the closest relative in the database. The

retrieved sequences ranged from 86% to 96% identity to arsC

sequences available in GeneBank.

Dissimilatory arsenate reductase arrA genesThree primer sets were used for the amplification of the As

respiratory gene arrA. The first primer set arrA1 included the arrAf

and arrAr primers [6] that amplify a ,160–200 bp fragment of

the gene. The optimized PCR conditions included incubation at

95uC for 10 min, followed by 40 cycles of 95uC for 15 s, 50uC for

40 s, and 72uC for 1 min [6]. The concentration of each primer in

a single reaction was 0.3 mM.

The second primer set was arrA2, used in a nested PCR

approach with primers AS1f, AS1r and AS2f [24]. This primer set

was designed after comparison of conserved regions in the arrA

genes from Bacillus selenitireducens, Chrysiogenes arsenatis, Shewanella sp.

strain ANA-3, Desulfitobacterium hafniense DCB2, and Wolinella

succinogenes. The nested-PCR with the primer combination AS2f

and AS1r yielded a 625 bp product. The first PCR step was

carried out by using a 5 min denaturation step at 94uC, followed

by 35 cycles of a 30 s denaturation at 94uC, primer annealing of 30

s at 50uC, and a 1min extension at 72uC. The second PCR

amplification (nested) was done with primers AS2F and AS1R

using the first PCR product as template. Nested PCR began with a

2 min denaturation step at 94uC, followed by 30 cycles of a 30 s

denaturation at 94uC, primer annealing of 30 s at 55uC, and a

1min extension at 72uC. The concentration of primers in each

PCR was 0.3 mM.

Finally, the third primer set for gene arrA was arrA3 (HAArrA-

D1f and HAArraA-G2r) that generated a 500 bp PCR product

[23]. The PCR conditions were initial denaturation at 95uC for 5

min, followed by 40 cycles of denaturation at 94uC for 30 s, primer

annealing at 53.5 uC for 30 s, and extension at 72uC for 30 s, with

an additional step at 85u C for 10 s, each primer at the

concentration of 0.3 mM.

Arsenite oxidation aioA geneThe PCR amplification of aioA was carried out using two sets of

degenerate primers (aioA1 and aioA2) (Table 2) to amplify

approximately 500 bp and bind at nucleotide positions 85–107

and the reverse primers bind at nucleotide positions 592–614 and

601–621, respectively, in the aioA sequence of Rhizobium sp. str.

NT26 [5]. The first set aioA1 consisted in aioA-1F and aioA-1R

and the PCR conditions were 95uC for 4 min followed by nine

cycles of 95uC for 45 s, 50uC (decreased by 0.5uC after each cycle)

for 45 s, 72uC for 50 s, followed by 25 cycles of 95uC for 45 s, 46uCfor 45 s, and 72uC for 50 s, and a final extension of 72uC for 5

min. The second set of primers aioA 2 were aioA-2F and aioA-2R,

and the PCR conditions for primer aioA2 were 90uC for 3 min,

followed by 40 cycles of 92uC for 1 min, 50uC for 1.5 min, 72uCfor 1 min and final extension of 72uC for 5 min. The concentration

of each primer was 0.3 mM.

Figure 2. Tree of the arsC genes used to select the sequences for primer design. The sequences targeted by each reported and designedprimer set are enclosed in colored rectangles. See Table 2 for primer sequences. The tree was constructed by Neighbor-joining. Bootstrap values for500 replicates are indicated at the nodes.doi:10.1371/journal.pone.0078890.g002



Arsenite transport genes acr3The amplification of acr3-1 and acr3-2 genes were done with two

pairs of degenerate primers (0.3 mM), dacr1F and dacr1R for acr3-

1 and dacr5F and dacr4R for acr3-2 as described by Achour et al.

(2007) [29]. The cycling conditions for all PCRs consisted of 5 min

of denaturation at 94uC followed by 35 cycles of 45 s of

denaturation at 94uC, 45 s of annealing at 57u–52uC with a 0.5uCdecrement per cycle during the first 10 cycles, and 30 s of primer

extension at 72uC. This was followed by an extension reaction at

72uC for 7 min.

The PCR ingredients for all primer sets included 10% (v/v)

DMSO and 0.5U of Taq DNA Polymerase (Invitrogen, Tech-

Line). The presence of PCR amplification products was verified by

electrophoresis in 1% agarose gels stained with ethidium bromide.

The DNA sample reference 595 obtained from a coastal marine

environment in Blanes Bay Microbial Observatory was used as a

negative control. Fusibacter sp. 3D3RP8 (FR873490) obtained in

those studies was used as positive control for reduction and

negative control for oxidation, and Herminimonas arsenicoxidans was

used as negative control for reduction and positive control for

oxidation.

Denaturing gradient gel electrophoresis (DGGE)The extracted genomic DNA of 16 of the samples was used as

target in the PCR to amplify 16S rRNA genes. Bacterial fragments

were amplified with the primer set 358FGC-907R [43] and

DGGE was performed as described previously [43]. The bands

were carefully excised from the DGGE gel with a razor blade

under UV radiation for further identification as has been reported

before [46]. Sequencing was carried out directly on PCR products

with the 341F primer in Macrogen. Sequences were deposited in

GenBank (accession numbers AB844273-AB844320).

The identity and similarity to the nearest neighbor of DGGE

band sequences were obtained using the BLAST algorithm [45].

The relative abundance of the major groups was calculated adding

the number of the DGGE bands with sequences affiliated to those

groups after the phylogenetic analysis.

Construction of 16S clone librariesSamples P9 (water) and P4 (sediment) from Salar de Ascotan

were selected for clone libraries because they had some the

lowest (3.4 mg L21) and the highest (1,210 mg Kg21) total

arsenic concentrations, respectively. 16S rRNA genes were

amplified by PCR with the universal primers 27F Mod (59-

AGR(AG) GTT TGA TCM(AC) TGG CTC AG-39) and 1492R

Mod (59-GGY(CT) TAC CTT GTT AY G ACT T-39) and

cloned into pCR2.1 vector with the TOPO TA cloning kit

Catalog #4500-01 (Invitrogen Carlsbad, California) and trans-

formed into TOP10 chemically competent cells. The trans-

formed cells were grown on Luria-Bertani plates containing

50 mg of Kanamycin mL21, 20 mg of 5-bromo-4-chloro-3-

indolyl-b-D-galactopyranoside (XGal) m L21, as recommended

by the manufacturer and incubated overnight at 37uC. Cloning

and sequencing were carried out as previously described [43].

Clones were analyzed with database nucleotide BLAST against

Nucleotide collection (nr/nt). Operational Taxonomic Units

(OTUs) were determined by Restriction Fragments Length

Polimorphism (RFLP) with enzyme HAE III. All sequences

belonging to the same OTU were found to be .97% identical.

Sequences were deposited in GenBank under accession numbers

FM879025 to FM879135.

Results

Geographical location, physicochemical data and biological

parameters for all sites sampled are shown in Table 1. The

sampling spots from Salar de Ascotan are shown in Figure 1 B.

The samples were obtained at different years and seasons and the

temperatures ranged between 0uC and 25uC, except for samples

from El Tatio geyser field, with temperatures ranging between

52uC and 78C. Salinity values were between 0.1 and 309 g L21,

conductivity ranged between 0.3 and 193.4 mS cm21 and total

dissolved solids (TDS) between 143 and 628000 mg L21. Sample

P6, collected on August 9, 2005, consisted of brine with the highest

values of salinity, conductivity, and TDS. Dissolved oxygen ranged

between 5.0 and 10.8, except for samples from El Tatio geyser

field SL1 and SL2 (DO 2.8 – 1.5, respectively). We found total

arsenic concentration values between 0.04 and 212 mg L21 in

water samples, and between 370 and 9440 mg Kg21 in the

sediments. These are some of the highest values of total arsenic

reported so far [1]. The concentrations of DAPI-stained cells

ranged between 1.66105 and 4.36107 cells mL21 in the water

samples and between 5.86105 and 1.96108 cells g21 in the

sediments (Table 1).

The relative abundance of the major bacterial groups was

analyzed by DGGE (Fig.3, Table S1). There was considerable

variation among the 16 samples (indicated by asterisks in Table 1).

For example, one sample was dominated by Bacteroidetes and

another by Deinococcus-Thermus, even though neither one of this

groups was abundant in general. Overall, however, Proteobacteria

and Firmicutes were the dominant groups (Fig. 3, Table S1). Two

samples, one water with low As and one sediment with high As,

were selected to build clone libraries as examples (Fig. 4).

Firmicutes were clearly dominant in both, followed by Proteo-

bacteria. The particular Proteobacterial classes in each one,

however, were different: gamma and epsilon in the water sample

and alpha, gamma and delta in the sediment.

Distribution of genes involved in As redoxtransformations and transport

We used a total of 11 primer combinations to explore the

presence of As related genes in 19 water and 10 sediment samples

from Salar de Ascotan, El Tatio geyser field, and two desert

streams in Atacama (Fig. 1, Table 1).

Primers for the arrA gene retrieved amplicons from all samples

(Fig. 5). In most cases, the three primer pairs tested produced

amplicons. In the water samples, primers arrA1 and arrA2 always

produced the same result (17 positives and two negatives). In the

sediment samples, however, primer arrA1 was always positive

while arrA2 had three negative results. Primer pair arrA3 was the

one with the largest number of negatives (three water and four

sediment samples) but in two water samples it was the only one

with positive results. In summary, all samples were positive with at

least one of the primer sets.

The results for the arsC gene were more complicated (Fig. 5).

We tried three primer pairs from the literature and tested three

new ones designed in the present study. Primer pair arsC-Grx-Sun

targeted the genes of Enterobacteria, Pseudomonas fluorescens and

Acidiphilum multivorum (Fig. 2), and it produced positive results only

in the six water samples with the lowest As concentrations. Primer

pair arsC-Grx-Saltikov (specific for Shewanella) did not produce

clear results. Four samples showed a very faint band in the gels

that was difficult to consider as positive. In one case, two bands

appeared suggesting there was a non-specific amplification.

Finally, there was one clear band in two more samples: the water

sample with the lowest As concentration and the P4-04 sediment



sample. In view of these ambiguous results, we cannot trust this

primer pair. Primer pair arsC-Trx-Villegas was designed to target

a group of Firmicutes genes and it was positive only in water

sample P9. This sample produced amplicons with all the primer

sets tested (Fig. 5).

The existence of arsC genes not targeted by the three primer sets

described so far was a likely explanation for their failure to retrieve

genes in Ascotan [19]. The phylogenetic tree built with the arsC

sequences obtained from the completely sequenced genomes

confirmed the occurrence of a third prokaryotic cluster of arsC

sequences, in addition to the two prokaryotic clades proposed by

Mukhopadhyay et al. (2002) [12]. The third cluster consisted

mostly of Firmicutes, but was closer to the enterobacterial GRX

than to the Firmicutes TRX cluster (Fig. 2).

The designed primer pairs arsC-Trx1a and arsC-Trx1b

targeted essentially the same groups as arsC-Trx-Villegas (Fig. 2)

which produced amplicons only in one sample (Fig. 5). However,

we obtained positive amplification in most samples, except in two

water samples (V-10_2006 and P6-05) and one sediment sample

(P4-04), for the primers arsC-Trx1a. The primer set arsC-Trx1b

was positive in five water and four sediment samples, one of each

had not amplified with primer set arsC-Trx1a. The arsC-Trx1a

and arsC-Trx1b sets designed to match with the first Firmicutes

group were able to retrieve genes from most of the samples,

Figure 3. Relative abundance of different phylogenetic groups of bacteria in 16 (indicated by an asterisk in Table 1) of the samplesanalyzed by DGGE.doi:10.1371/journal.pone.0078890.g003

Figure 4. Relative abundance of major taxonomic groups in clone libraries from two selected samples (P9 water and P4 sediment)with different total arsenic concentrations from Salar de Ascotan.doi:10.1371/journal.pone.0078890.g004



including the ones with the higher As concentrations. However,

when we used the primer set arsC-Trx2 designed to match the

other Firmicutes group only nine water and eight sediment

samples, spanning different arsenic concentrations, amplified. The

samples with positive results using the primer set arsC-Trx2 also

gave positive PCR with primers arsC-Trx1a and arsC-Trx1b.

Overall, these results are consistent with Firmicutes being

dominant in these samples (Figs. 3 and 4). Likewise, Firmicutes

were the most common bacteria found in enrichment cultures

from these samples [46]. In total, 86% of the samples showed

positive PCR results with arr1 primers and arsC primers designed

for the two Firmicutes clusters (Fig. 5).

In summary, the Enterobacteria related arsC genes were present

in the water samples with the lowest As concentrations only, while

the Firmicutes related genes could be found in samples spanning

the whole range of arsenic concentrations evaluated in this study,

although not in all the samples.

The fragments of arsC genes retrieved after sequencing of the

PCR product of sample P9 using the primer pair arsC-Grx-Sun

formed a clade within the Enterobacteria (Fig. 2). Meanwhile the

sequences of the fragments obtained after the PCR of 10 samples

2P405, P11, P604, P704, P806 P405, P604, QAB, P9 QJere2

using the primer pair arsC-Trx1a and arsC-Trx1b were found

within the Trx clade described before [12] (Fig. 2).

We tested two primer pairs targeting the aioA1 and aioA2 genes

respectively. aioA2 was amplified from most samples (fifteen water

and seven sediments), while aioA1 appeared in a subset of the

samples positive for aioA2. Seven samples with very different As

concentration did not produce any amplification. Finally, two

primer sets were tested targeting the acr3 As(III) transporter gene.

acr3 2 showed positive results in seven water and five sediment

samples, while acr3 1 only had one positive result.

Discussion

As(V) dissimilatory reductionThe capability to respire As(V) is widespread in different

prokaryotic groups such as Proteobacteria, Firmicutes, Chrysio-

genetes, Deinococcus-Thermus, Deferribacteres and Chrenarch-

aeota. Yet, all well studied As(V) respirers use homologs of the

Figure 5. Total [As] in the natural environments analyzed (black triangles for water samples [mg/L] and yellow triangles forsediment samples [mg/Kg]), and range of concentrations where the arsenic related genes could be found with different primer sets(Table 2). The presence of the different genes is indicated by shades of gray; absence by white.doi:10.1371/journal.pone.0078890.g005



arrAB operon for this process. The arrA gene codes a respiratory

arsenate reductase and arrB transfers electrons from the electron

transport chain to the previous enzyme, thus obtaining energy

from the electron transport. The ArrA proteins form a well-

defined cluster within the family of DMSO reductases [5,6]. Three

different primer sets have been developed targeting the arrA gene.

Malasarn et al. (2004) [6] developed the arrA1 set matching a

diverse group of eight bacteria including Proteobacteria, Chrysio-

genetes and Firmicutes (Table 2). With this primer set, these

authors were able to retrieve arrA genes from Haiwee Reservoir [6]

and Hollibaugh et al. (2006) [47] found the gene in Mono Lake.

Lear et al. (2007) [24] used primer set arrA2 to detect the genes in

mesocosms with sediments from contaminated aquifers. Finally,

Kulp et al. (2006) [23] modified the arrA1 primers developing

primer set arrA3 to include archaeal genes and retrieved

sequences from Mono and Searles Lakes anoxic sediments. The

latter primers produced longer amplicons and targeted a larger

number of arrA genes than the arrA1 set (Table 2). We used the

three primer sets trying to maximize the recovery of arrA genes.

Surprisingly, primer set arrA3 had more negative results than

arrA1 (Fig. S2), despite the fact that it was supposedly an

improvement over the latter. These results could be understood if

there are still many arrA genes in nature that have not yet been

retrieved in sequence collections. Thus, the primers developed

with just a few pure cultures are likely missing different arrA genes

present in the environment.

At any rate, the interesting fact was that arrA genes were present

in all samples (Fig. S2). A priori, we had expected this gene to be

present at the higher end of the spectrum of As concentrations

only, since dissimilatory reduction requires large concentrations of

the electron acceptor [48]. Also, we were expecting it in the

sediment samples where anaerobiosis is more likely. Yet, the gene

was present throughout a range of six orders of magnitude of As

concentrations and in all water samples with dissolved oxygen

concentrations close to saturation. When most probable number

estimates of arsenate reducing bacteria were carried out in a

previous study, positive growth was found in all the sediment

samples with high As concentrations (larger than 370 mg Kg21),

while all the water samples with As concentrations lower than

4 mg L21 showed negative results [22]. At intermediate concen-

trations, some samples were positive and others negative. These

results are consistent with As-respiring bacteria being present only

at the highest As concentrations, particularly in the sediments.

One possible explanation for the presence of this gene in aerobic

environments is the recent discovery that the respiratory arsenate

reductase may act in some cases as a bidirectional enzyme and the

redox potential of the molybdenum center could play a role in

determining the direction [26,49,50]. The expression of the related

arxA gene (not targeted in this study) is only induced with arsenite

and under anaerobic conditions [50]. Thus, the enzyme could be

used in different ways in different environments. In addition,

evidence has been reported of a wide distribution of anaerobic

arsenite oxidation activity associated to arx genes (arx-based

arsenotrophy) [26]. Further, the arx gene resembles the genes

encoding the catalytic subunit of the ArrA [49] and ArxA has been

established as a new clade of arsenite oxidases within the DMSO

reductase family of molybdenum oxidoreductases [26]. However,

the primers used to amplify arrA gene hit the arxA gene sequence

of Alkalilimnicola ehrlichii MLHE-1 and only the forward primer

used to amplify arrA gene partially hits the arxA gene sequence of

Ectothiorhodospira sp. PHS-1.Therefore, a cross reaction is likely in

view of the available arxA sequences in data bases.

As(V) detoxifying reductionThe primers targeting the GRX class including enterobacterial

gamma-Proteobacteria and Shewanella [19,51] and the TRX class

[20] (Table 2) retrieved few arsC genes from our samples (Fig. 5

and Fig. S2). arsC-Grx-Sun primers only found targets in the six

water samples with the lowest As concentration and the arsC-Grx-

Saltikov primers gave doubtful results. This is not surprising since

the last pair was designed specifically for Shewanella and there is no

reason why this bacterium should be present in Ascotan. Bacteria

with arsC genes of the Grx-enterobacterial clade do not seem to

tolerate As concentrations above 4 mg L21. In agreement with

this, Sun et al. (2004) [19] only found the arsC gene using general

PCR after incubation, and they only detected it by real-time PCR

in the natural samples from a contaminated mine soil. As

concentrations in the studied sites were not reported, however,

and comparison with ours is not possible. Likewise, the gene was

not detected in a Cambodian contaminated sediment with

13.1 mg Kg21 of arsenic [24]. These primers may not be the

most efficient at retrieving arsC genes from different environ-

ments, likely because they target only a small group of bacteria.

Obviously, the diversity of known arsC sequences restricts the

detection of arsC genes when using probes designed from

phylogenetically different organisms [19].

We were more intrigued by the results from the arsC-Trx-

Villegas set. This primer set was designed targeting a group of arsC

genes coding TRX-coupled enzymes [20], mostly from Firmicutes

(Table 2). In the present study, only one sample was positive with

this primer set (Fig. S2). This was unexpected because the DGGE

and the clone library of the 16S rRNA gene from samples showed

Firmicutes to be dominant members of the community (Figs. 3 and

4).

Jackson and Dugas (2003) [17] analyzed a larger data set of arsC

sequences than that considered by Mulkophadhyay et al. (2002)

[12] and concluded that the most likely explanation for the

diversity of arsC genes was an early origin of the gene with

diversification leading to the three main branches proposed by

Mulkophadhyay et al. [12]. Several other branches with a small

number of known sequences in the data bases are part of that

diversity [12]. Their tree, in effect, showed several different clades

in addition to the three proposed by Mulkophadhyay et al. (2002).

Moreover, recently, a new family of arsC genes (the mycothiol

(MSH)/mycoredoxin (Mrx)-dependent class) has been discovered

in Corynebacterium glutanicum [14] and Mycobacterium tuberculosis [15].

Both the number and type (Trx or Grx) of arsC genes present in

the genomes of prokaryotic organisms have been shown to impact

their arsenic resistance level [16,52–54]. The Trx reducing system

has been reported to be the most efficient in arsenate decontam-

ination [16]. In addition, many Trx-linked arsenate reductases

have been found in low G + C Gram positive bacteria [7] and we

have shown that these bacterial groups are predominant in arsenic

impacted environments [46,55]. Thus, the absence of Grx-

associated arsC genes in the environments with intermediate and

high concentrations of As found in the present study could be

explained by the predicted lower efficiency of the reductases.

As(III) oxidationA large variety of genes involved in As(III) oxidation have been

described [10,56]. Lett et al. (2011) [25] attempted to unify the

nomenclature, since many homologous genes had been named

differently by different authors. We have followed their nomen-

clature here. The genes include two subunits of the arsenite

oxidase enzyme (aioA and aioB), a sensor histidine kinase (aioS), a

transcriptional regulator (aioR) and an oxyanion binding protein

(aioX). Inskeep et al. (2007) [5] developed two sets of primers



targeting the aioA gene (Table 2). They used seven phylogenetically

distant bacteria for the design and tested the primers with a series

of known As oxidizer pure cultures. These primer sets were then

successfully used to retrieve aioA genes from different environ-

ments, including contaminated soils [57], geothermal springs [5]

and a lake sediment. The As concentrations of these environments

ranged between 0.01 and 1.74 mg L21 As, at the lowermost end of

the range of values considered in the present study. These primers

were used to detect the presence of the gene in a number of pure

cultures [57] finding the aioA gene only in five Proteobacteria out

of 58 As-resistant bacteria tested. Campos et al. (2011) [58] also

found this gene in bacteria isolated from Quebrada Camarones, a

desert stream a few hundred kilometers from our studied sites. To

our knowledge, these primers have not been tested in other natural

environments.

Using these primers, we found 14 and 22 positive responses

from primer sets aioA1 and aioA2, respectively (Fig. S2). When

primer set aioA1 gave a positive response, the other set also gave a

positive response. The positive amplifications were distributed

throughout the range of As concentrations and both in water and

sediment samples. This extends the range of concentrations where

this gene has been found in environmental samples by four orders

of magnitude [33]. Most of the samples that did not show the

presence of this gene were from hot springs, both water and

sediment samples, but we do not have enough data to test whether

this is a robust characteristic or not.

As(III) transportThere are two unrelated families of arsenite transporters in

Bacteria. The arsB pump is associated with the arsC reductase. We

decided not to look for the arsB transporter because it is most

frequently associated with arsC, a gene we were already targeting it

with six primer sets. We assumed a distribution similar to that of

the arsC genes (see above). The last As related genes we tested were

the As(III) transport genes acr3, of which two varieties have been

identified. Achour et al. (2007) [29] designed two sets of primers

acr3 1 and acr3 2 for those varieties and tested these primers plus a

set of primers for the arsB gene with a series of As-resistant isolates

from soil samples with different levels of arsenic contamination.

They found that the acr3 gene was more typical than arsB in those

41 strains. In addition, arsB was prevalent in Firmicutes and

Gammaproteobacteria while acr3-1 and acr3-2 genes were more

common in Actinobacteria and Alphaproteobacteria, respectively.

Cai et al. (2009) [57] successfully used these primers to detect the

genes in As-resistant bacteria isolated from contaminated soils.

The acr3-1 gene was present in 10 Gammaproteobacteria and two

Actinobacteria, while the acr3-2 gene was found in 21 isolates from

Alpha-, Beta- and Gammaproteobacteria, seven in each. These

primers with modifications have also been tested in samples taken

from Rifle aquifer CO, USA, with As levels of 1.5 mM, and

sequences from both subfamilies were recovered [59] With the

acr3 1 primers we found amplification only in one sample from a

hot spring at El Tatio. This result is consistent with the absence of

Actinobacteria in water and sediment samples from Ascotan

according to the 16S rRNA DGGE and gene library data (Figs. 3

and 4). With the acr3 2 set, however, we obtained amplification

from seven water and five sediment samples (Fig. S2). Interest-

ingly, the amplification results with the arsC-Trx2 and the acr3 2

sets of primers were coincident in more than 60% of the samples.

This makes sense considering that the genomes of all the

microorganisms used to design the arsC-Trx2 primers also

contained the acr3 gene, while this was not the case of the

microorganisms used to design the arsC-Trx1a and arsC-Trx1b

primer sets.

An increased number of positive amplification of As related

genes was found in sediment samples compared to water samples.

The analysis of the 16S rRNA genetic libraries from a sediment

and a water sample (Fig. 4) indicated an SChao1 index calculation

of 590 for sediments and 42.5 for water samples (at 99%

similarity). This higher diversity in sediment than in water samples

has been found in similar environments [60-62]. Thus, the

increased number of As processing genes in the sediments could be

a consequence of the larger phylogenetic diversity of the sediment

communities.

Concluding remarksMost As-related genes were widely distributed throughout the

six orders of magnitude range of As concentrations obtained after

analyzing very heterogeneous environments, such as shallow

lagoons brines, sediments, hot springs, and salt deposits. This study

is based on PCR and some concerns should be considered. Instead

of using specific PCR primers with a given sequence, we tried to

increase the primer coverage by using degenerate PCR primers,

designed after an extensive GenBank search, to target the

sequences present in such diverse environmental set. We were

very careful to minimize the limitations to properly obtain specific

PCR products. Priming conditions were optimized after testing the

annealing temperatures coupled to amplicon cloning and

sequencing. To what extent mismatches may have affected the

efficiency of the PCR is however something unknown.The fact

that some genes could not be amplified in some of the samples

could be due to the environmental heterogeneity or to method-

ological limitations. The arsC gene, however, provided a rather

clear pattern. The enterobacterial related variant of the gene was

only present in the lower As concentrations, while the Firmicutes

related gene variants were present in many different samples

including those with the highest As concentrations. The arsC gene

has experienced a large diversification, and apparently different

bacterial lineages have inherited variants of the gene providing

different degrees of protection against arsenic. The diversity of As

processing genes found in the sediment samples was larger than in

the water samples, in agreement with the larger phylogenetic

bacterial diversity found in sediments than in water samples.

Supporting Information

Figure S1 Primer targeting location on the arsenate cytoplasmic

reductase genes (arsC) from Firmicutes phylum. A) Primer set

arsC-Trx1a (black frames) and b (grey frames) and arsC sequences

from Bacillus thuringiensis, Bacillus cereus, Geobacillus kaustophilus,

Bacillus clausii, Oceanobacillus iheyensis and Bacillus halodurans; B)

Primer set arsC-Trx2 (black frames) and arsC sequences from

Geobacillus thermoglucosidasius, Geobacillus sp., Alkaliphilus oremlandii

OhILAs and Bacillus subtilis.

(TIF)

Figure S2 Number of samples with PCR positive results for

targeted genes using the previously described and newly designed

sets of primers (Table 2).

(TIF)

Table S1 Blast output for sequenced DGGE bands from water

an sediment samples in Salar de Ascotan, Tatio Geyser field and

Salar de Atacama (Quebradas Aguas Blancas and Jere).

(PDF)

Acknowledgments

We thank Vanessa Balague, Mauricio Acosta and Dayana Arias for

technical support.



Author Contributions

Conceived and designed the experiments: LE EOC GCH CPA CD.

Performed the experiments: LE. Analyzed the data: LE EOC GCH CPA

CD. Contributed reagents/materials/analysis tools: LE CPA CD. Wrote

the paper: LE CPA CD GCH. Description of the geological setting of the

basins for designing the field campaigns and to understand the results

obtained: GCH.

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